A greater understanding of the epigenetic processes that contribute to γ‑globin silencing will ultimately help inform the development of therapeutic strategies for the reactivation of γ-globin. In this study, we generated a murine erythroleukaemia cell line that carries an intact 183kb human β-globin locus modified to express eGFP under the control of the γ-globin promoter (GG-MEL). The GG-MEL reporter was used to screen a retroviral shRNA library containing 1027 shRNAs targeting 158 genes encoding epigenetic regulators. Following retroviral transduction, GFP-positive and GFP-negative samples were collected over the course of differentiation. Next generation sequencing and bioinformatics analysis were used to compare differences in shRNA representation between the GFP-positive and GFP-negative samples. The screen identified several genes that are known regulators of γ-globin silencing, as well as genes that have not previously been associated with γ‑globin regulation. Following validation of novel candidates, Kdm2b emerged as the top regulator of γ-globin silencing.
Molecular characterisation was conducted in parental MEL cells and included RNA-seq to investigate global changes in gene expression upon Kdm2b knockdown, and ChIP-seq to identify global binding sites of KDM2B. ChIP-seq analysis revealed strong enrichment of KDM2B at actively expressed β‑globin genes, but was absent at the developmentally silenced embryonic/fetal globin genes. Knockdown of Kdm2b resulted in increased expression of the γ-globin reporter in primary erythroid cells derived from the GG murine fetal livers. These results suggest Kdm2b may be an activator of β-globin gene expression and down regulates γ-globin through a competitive model.
More recently, KDM2B has been identified to be a component of the non-canonical polycomb repressive complex PRC1.1, which is associated with activating histone modifications. We propose that KDM2B regulates the β‑globin gene through interaction with non-canonical PRC1.1 complex and protects the gene from DNA hypermethylation.